Dear flowers, As there were so many people asking for the CFSE summary, I am just posting it on the list. Hope no none objects. Summary: CFSE on CB cells Thank you: Adrian - Doug - Julie - Sathi - Usha for the advice Clumping of thawed cells - lower CFSE concentration - Dnase (50U/mL) - FCS (2-5%) (if very low cell number) - More with whole CB or MNC, less with CD34+ enriched fraction Compensation - (with FL-2): use bright FL-2 stain and reduce FL-2 detector amplification - FL-2-FL-1 compensation: 90-100% - Titration of all PE antibodies - (with FL-3): gating out of dead cells with TOPRO-3 instead of PI - lower CFSE concentration (esp if analysis shorter than 24H after labeling) - keep labeled cells in culture for 24H (overnight) before analysing them (this allows stablisation of cell labeling after initial quenching of some dye out of the cells) - Aim initial peak fluorescence at about 103 on FL-1 scale Freezing of CFSE in DMSO at -20°C - kan be kept indefinetely / 2 years (?), but intensity lowers with every thaw Kind regards, Tessa Tessa Kerre, MD Gent University Hospital Belgium
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